Development of novel sensor platforms for sensitively and specifically detecting small molecules in solutions is very important for monitoring of disease-related metabolites, environmental pollutants and food toxins. Low-molecular-weight materials, such as metabolites, environmental pollutants and food toxins, have generally been analyzed by complex techniques, such as GC/MS or HPLC, which are time-consuming even by skilled workers and cannot be applied to on-site analysis (Stales, C. A. et al, Environ. Toxicol. Chem., 20: 2450, 2001). In order to succeed in on-site real-time detection, small-scale systems and specific binding reagents have been required.
As portable platforms for detecting small-molecular-weight materials, detection platforms such as surface plasmon resonance platforms are known, but it is known that it is difficult for these platforms to detect materials having a very low molecular weight of 400 or less. In addition, it was reported that, when analysis was conducted using an aptamer specifically binding to 17-β-estradiol (MW: 272) at the concentration of 1 μM (272 ppb), analysis for low-molecular-weight materials as described above is impracticable as shown in FIG. 1 (Kim et al., Biosens. Bioelecrtron., 22:2525, 2007). Thus, development of novel platforms capable of detecting even low-molecular-weight materials in a specific and sensitive manner has been demanded.
Also, for specific and sensitive detection of analytes, reagents specifically binding to analytes such as antibodies are required. However, low-molecular-weight materials are generally too small to generate antibodies in animals or are strongly toxic such that they cannot produce antibodies. For this reason, novel specific binding reagents targeting small molecules have been demanded.
Meanwhile, the term “aptamer” as used herein refers to a single-stranded oligonucleotide (single-stranded DNA or RNA molecule) that can bind specifically to its target with high affinity. The aptamer can be used as a biosensor element capable of binding to a molecule in a detection/analysis system, and thus has been recognized as a substitutive for antibody. Particularly, the aptamers can be used as molecules targeting various organic and inorganic materials, including toxins, unlike antibodies, and once an aptamer binding specifically to a certain material is isolated, it can be consistently reproduced at low costs using automated oligomer synthesis methods. Since an aptamer-based biosensor of measuring a target protein using a fluorescence-labeled aptamer was first developed in 1996, various aptamer biosensors have been developed based on the advantages and structural properties of the aptamer (Yeon-Seok KIM & Man-Bock GU, NICE, 26(6): 690, 2008).
However, prior analysis methods employing aptamers were competitive analysis methods in an aptamer attached to a specific material is detached or allowed to bind to other materials. Thus, development of a method of using an aptamer to detect a material in a more direct and simple manner has been required.
Meanwhile, carbon nanotube (CNT)-based biosensors are highly attractive portable platforms for detecting small-molecular-weight materials. Single-wall carbon nanotube (swCNT)-field effect transistors (FETs) were useful as small-scale sensors for highly sensitively detecting chemical materials, compared to other detection platforms, such as quartz-crystal microbalance sensors, electrochemical impedance spectrometry sensors, surface plasmon resonance sensors and light-addressable potentiometric sensors (LAPSs) (Kim, T. K. et al., Advanced Materials, 20:1, 2008; Kong, J. et al., Science, 287:622, 2000; Snow, E. S. & Perkins, F. K., Nano. Lett., 5:2414, 2005).
However, recent swCNT-FET for detecting small molecules have been limited only to a gas or vapor phase rather than a liquid phase containing most biological metabolites or toxins of interest. In addition, because detection of non-polar small molecules in solutions was not within the detection range of FET-based sensors, it was difficult to detect small molecules using the FET-based sensors (Heller, I. et al, Nano. Lett., 8:591, 2008).
For specific and sensitive detection of analytes, swCNT-FET sensors are required to have specific binding reagents such as antibodies as functional groups, and small molecules are generally too small to generate antibodies in animals or are strongly toxic such that they cannot produce antibodies.
Thus, the present inventors have made extensive efforts to provide a novel detection method capable of detecting even a small molecule, particularly in a solution, and, as a result, have found that, when a sample and a second aptamer are added to a first aptamer immobilized on a solid phase so as to form a bond sandwiched between the first aptamer, the target material and the second aptamer, even a non-polar low-molecular-weight material such as bisphenol A can then be detected, thereby completing the present invention.
The present inventors have also found that even a non-polar low-molecular-weight material present at the pM level can be detected by immobilizing a first aptamer as a probe on an FET sensor, adding a sample and a second aptamer thereto, and then measuring a change in electric current in the FET sensor, thereby completing the present invention.
The present inventors have also found that even a non-polar low-molecular-weight material can be detected by immobilizing an aptamer as a probe on an AAO sensor, adding a sample thereto, and then measuring a change in capacitance in the AAO sensor, thereby completing the present invention.